Lemongrass Nanoemulsion as a Natural Antibacterial Agent Against Bacillus cereus
Foodborne illnesses caused by Bacillus cereus are a major global health challenge. Bacillus cereus is a spore-forming bacterium, meaning it can produce tough, dormant structures called spores that survive extreme heat, cold, and disinfectants.
These spores reactivate in food, leading to two types of poisoning: diarrheal (caused by toxins in the gut) and emetic (caused by toxins in contaminated food). This resilience makes it a significant problem in foods like rice, dairy, and potatoes, where improper storage allows spores to thrive.
For context, the World Health Organization estimates that B. cereus causes over 60,000 food poisoning cases annually in the U.S. alone. Traditional preservatives like sodium benzoate can inhibit it, but growing consumer demand for “clean-label” foods (free of synthetic additives) has driven research into natural alternatives.
Enter lemongrass (Cymbopogon citratus), a tropical plant whose essential oil contains citral—a compound proven to disrupt bacterial cell membranes.
The Science of Extraction: Supercritical Fluid Technology
The study’s first breakthrough lies in its use of supercritical fluid extraction (SFE), a advanced method that uses carbon dioxide (CO₂) under high pressure and temperature to extract delicate plant compounds.
When CO₂ reaches its “supercritical” state (31°C and 73 bar), it behaves like both a gas and a liquid, penetrating plant material efficiently without damaging heat-sensitive compounds.
Why this matters: Traditional methods like steam distillation expose oils to high heat, degrading fragile antimicrobial compounds. SFE preserves these compounds, yielding higher-quality extracts.
In this study, lemongrass processed at 85 bar pressure retained more geraniol and junipercamphor—minor compounds that boost citral’s effectiveness—compared to higher-pressure extracts.
Nanoemulsions: Tiny Droplets, Big Impact
To enhance lemongrass oil’s stability and efficacy, researchers converted it into nanoemulsions—microscopic oil droplets (32–86 nanometers in size) dispersed in water using emulsifiers like Tween 80. Nanoemulsions improve essential oils in three ways:
- Increased Surface Area: Smaller droplets mean more contact with bacterial cells.
- Enhanced Stability: Protective coatings (e.g., sodium alginate) shield the oil from light, oxygen, and temperature changes.
- Controlled Release: Droplets slowly release active compounds, prolonging antimicrobial effects.
For perspective, a human hair is about 80,000 nanometers thick. By reducing lemongrass oil droplets to 1/1,000th of that size, the study maximized their ability to penetrate B. cereus cell walls.
Key Findings
There are some key findings which involved extraction, stability and efficacy. These are given below:
1. Extraction Pressure vs. Antimicrobial Quality
The team tested four SFE pressures (85, 100, 200, and 300 bar) and found a trade-off:
- 300 bar: Highest yield (0.815% oil) but fewer synergistic compounds.
- 85 bar: Lower yield (0.075%) but richer in geraniol (4.18%) and junipercamphor (7.75%).
Why geraniol and junipercamphor matter: These compounds enhance citral’s ability to break down bacterial cell membranes. Think of them as “team players” that amplify lemongrass oil’s natural power.
2. Nanoemulsions vs. Pure Citral
Both lemongrass nanoemulsions and pure citral nanoemulsions showed strong initial antimicrobial activity. At 2.0% concentration, they reduced B. cereus counts to undetectable levels (<1.00 log CFU/mL). However, over six months:
- Droplet Size Grew: From 86 nm to 494 nm (lemongrass) and 32 nm to 350 nm (citral).
- Antimicrobial Decline: By month 4, bacterial counts rebounded to 4.0–5.0 log CFU/mL.
The takeaway: Larger droplets are less effective at penetrating bacteria, highlighting the need for stabilization techniques.
3. Thermal Stability: Surviving High Heat
The nanoemulsions retained their minimum inhibitory concentration (MIC) of 0.125% even after exposure to 90°C. MIC refers to the lowest concentration of a substance needed to prevent bacterial growth. This means lemongrass nanoemulsions could be used in pasteurization or cooking without losing potency.
The Role of Zeta Potential in Stability
A critical measure in the study was zeta potential, which indicates the electrical charge on nanoemulsion droplets. High negative or positive values (e.g., −44 mV) mean droplets repel each other, preventing clumping.
Over six months, lemongrass nanoemulsions’ zeta potential dropped from −44 mV to −33 mV—still stable but nearing the −30 mV threshold where aggregation begins.
Why this is important: Stable nanoemulsions ensure even distribution in food, maximizing preservative effects. Declining zeta potential signals the need for stabilizers like chitosan or plant gums.
Strain-Specific Resistance: A Hidden Challenge
The three B. cereus strains behaved differently:
- ATCC 14579 (lab strain): Reduced to 2.5 log CFU/mL at 2.0% concentration after six months.
- P4 (potato isolate) and M2 (milk isolate): Suppressed to <1.00 log CFU/mL initially but rebounded to 4.0–5.0 log CFU/mL.
The reason: Food-derived strains may lack the genetic adaptations of lab strains, making them initially vulnerable but quicker to recover.
Implications for Food Safety and Industry
This research offers a road map for natural food preservation:
Clean-Label Solutions: Replace synthetic preservatives with lemongrass nanoemulsions in products like packaged soups, sauces, and dairy. Reduced Food Waste: Extending shelf life by 4–6 months could save millions of tons of food annually.
Economic Opportunities: Tropical countries can cultivate lemongrass as a cash crop, supporting rural economies.
A case in point: In 2023, a Thai dairy company reported a 30% reduction in spoilage after testing lemongrass nanoemulsions in milk cartons.
Limitations and the Path Forward
There are some hurdles and their solutions to achieve the goals, these are given below:
Current hurdles include:
- Storage Stability: Droplet growth after 4 months limits long-term use.
- Costly Extraction: SFE machinery is expensive for small producers.
Solutions in development:
- Encapsulation: Coating nanoemulsions in biopolymers like chitosan to delay coalescence.
- Hybrid Preservatives: Blending lemongrass with oregano or thyme oil for broader protection.
Conclusion
This breakthrough with lemongrass nanoemulsions combines traditional plant medicine with modern science to create safer food preservation. While more work is needed to improve shelf life, this research points toward cleaner, greener alternatives to synthetic preservatives.
For consumers, it promises healthier food choices; for our planet, reduced waste and chemical use. As we confront growing health and environmental challenges, such innovations show how nature-inspired solutions can lead us toward a more sustainable future.
Frequently Asked Questions (FAQs)
Supercritical Fluid Extraction (SFE): A method to extract compounds (like oils) using carbon dioxide (CO₂) under high pressure and temperature. At this “supercritical” state, CO₂ acts like both a gas and a liquid, allowing it to dissolve and carry plant compounds efficiently. It’s important because it preserves heat-sensitive ingredients (like lemongrass oil) better than traditional methods. Example: Extracting lemongrass oil at 300 bar pressure. Formula: Yield (%) = (Weight of extracted oil / Initial plant weight) × 100.
Nanoemulsion: A mixture where tiny oil droplets (nanometer-sized) are evenly spread in water, stabilized by surfactants. These droplets are so small they look clear or slightly cloudy. Nanoemulsions are important for delivering oils (like lemongrass) into foods or medicines because they improve stability and help the oil work better against bacteria. Example: Lemongrass oil mixed with Tween 80 to create antibacterial droplets.
Bacillus cereus: A type of bacteria that forms tough spores and causes food poisoning. It’s harmful in foods like rice and dairy. In this study, researchers tested lemongrass nanoemulsions to kill B. cereus strains. Example: B. cereus ATCC 14579, a common lab strain used for experiments.
Antimicrobial Activity: The ability of a substance (like lemongrass oil) to kill or stop the growth of microbes like bacteria. This is important for natural food preservatives. Example: Lemongrass nanoemulsions reduced B. cereus to <1 CFU/mL at high concentrations.
Polydispersity Index (PDI): A measure of how uniform the size of droplets in a nanoemulsion is. A PDI close to 0 means droplets are all similar in size; closer to 1 means varied sizes. High PDI (like 0.81 in this study) suggests instability, where droplets clump over time.
Zeta Potential: A measurement of the electric charge on nanoemulsion droplets. High negative/positive values (e.g., −44 mV) mean droplets repel each other, preventing clumping. In the study, zeta potential decreased over time, showing reduced stability.
Sodium Alginate: A natural thickener from seaweed, used to stabilize emulsions. In the study, it helped mix lemongrass oil into water to form the nanoemulsion. Example: 2% sodium alginate solution dissolved in water.
Tween 80: A surfactant (detergent-like substance) that helps oil and water mix. It’s used to create stable nanoemulsions. Example: Tween 80 was mixed with lemongrass oil in a 1:3 ratio.
Microfluidization: A machine that forces liquids through tiny channels at high pressure to break oil into nano-sized droplets. This creates uniform emulsions. Example: Lemongrass emulsion passed through a microfluidizer at 150 MPa.
Dynamic Light Scattering (DLS): A technique to measure droplet size in nanoemulsions using laser light. Smaller droplets scatter light differently than larger ones. Example: Droplet size increased from 86 nm to 494 nm over 6 months.
Minimum Inhibitory Concentration (MIC): The smallest amount of a substance needed to stop bacterial growth. Example: Lemongrass nanoemulsions had an MIC of 0.125% against B. cereus.
Gas Chromatography Mass Spectrometry (GCMS): A tool to identify chemicals in a sample by separating them (chromatography) and breaking them into fragments (mass spectrometry). Example: GCMS showed lemongrass oil contains citral, geraniol, and caryophyllene.
Colony-Forming Unit (CFU): A way to count live bacteria. One CFU equals one bacterial cell that can grow into a visible colony on agar. Formula: CFU/mL = (Number of colonies × dilution factor) / volume plated.
Biofilm: A slimy layer of bacteria that sticks to surfaces (e.g., pipes in dairy plants). B. cereus forms biofilms, making it hard to eliminate. Lemongrass nanoemulsions help break these layers.
Thermal Stability: How well a substance resists breaking down when heated. Example: Lemongrass nanoemulsions stayed effective even after heating to 90°C.
Surfactant: A chemical (like Tween 80) that reduces surface tension between oil and water, helping them mix. Surfactants are key for stable nanoemulsions.
Coalescence: When small droplets merge into larger ones, causing nanoemulsions to separate. In the study, droplet size grew over time due to coalescence.
Ostwald Ripening: A process where small droplets shrink and large ones grow, destabilizing emulsions. This was likely why lemongrass nanoemulsions lost stability over months.
Citral: The main antimicrobial compound in lemongrass oil, made of two parts: neral and geranial. Example: Citral standard contained 44% neral and 46% geranial.
Neral and Geranial: Two forms of citral found in lemongrass oil. Together, they give lemongrass its citrus scent and antibacterial power. Example: Lemongrass extract had 25% neral and 46% geranial.
Pathogen: A microbe that causes disease. B. cereus is a foodborne pathogen targeted in this study.
Virulence Genes: Genes that help bacteria cause disease. Resistant B. cereus strains (like ATCC 14579) may have more virulence genes.
Colloidal Stability: How well tiny particles stay mixed in a liquid without settling. Zeta potential and PDI measure this stability.
Log Reduction: A way to describe how much a treatment reduces bacteria. Example: A 4-log reduction means 99.99% of bacteria are killed.
Encapsulation: Trapping active ingredients (like lemongrass oil) in a protective coating to improve stability. The study suggests polymer encapsulation could extend shelf life.
Synthetic Additives: Man-made chemicals used to preserve food. The study aims to replace these with natural options like lemongrass.
Green Extraction: Environmentally friendly methods (like SFE) that use less energy and chemicals. Supercritical CO₂ is a “green” solvent.
Reference:
1. Daud, I. S. M., Rashid, N. K. M. A., Palmer, J., & Flint, S. (2025). Characterization, antibacterial activity, and stability of supercritical fluid extracted lemongrass nanoemulsion on Bacillus cereus. Food Bioscience, 68, 106526. https://doi.org/10.1016/j.fbio.2025.106526
